Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Briciu-Burghina, Ciprian; Heery, Brendan; Regan, Fiona

doi:10.1039/c5an01021g

Cited by 35 publications

(20 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Its fluorophore 6-Chloro-4-Methyl-Umbelliferone (6-MUG) has a lower pKa value (6.12) than 4-MU and at pH 6.8 is almost fully dissociated into its highly fluorescent anionic form. Furthermore GUS catalysis rates for the two substrates are quite similar, Kcat ¼ 222 7 13.4 S 1 for 4-MUG and Kcat ¼ 207 7 8.5 S 1 for 6-CMUG at 37 °C and pH 6.8 [24].…”

Section: Introductionmentioning

confidence: 85%

“…Briefly the protocol involves two main steps: sample preparation and GUS quantification [24]. The sample was filtered through 0.45 μm syringe filters for bacteria capture and pre-concentration using 50 mL syringes, followed by lysing agent addition (100 mL).…”

Section: Environmental Sample Testingmentioning

confidence: 99%

“…Its fluorophore 4-Methyl-Umbellierone (4-MU) has a pKa of 7.8 and is highly fluorescent at pH values over 9 [8,9,13]. Recent work in our research group [24] demonstrates the use of 6-Chloro-4-Methyl-Umbelliferyl-β-D-Glucuronide (6CMUG) for continuous GUS assays with greatly reduced sample handling. Its fluorophore 6-Chloro-4-Methyl-Umbelliferone (6-MUG) has a lower pKa value (6.12) than 4-MU and at pH 6.8 is almost fully dissociated into its highly fluorescent anionic form.…”

Section: Introductionmentioning

confidence: 99%

“…The amount of fluorophore released in a certain period of time is directly proportional to the number of E. coli cells trapped. The assay performs optimally at 44 °C and at pH 6.8 [24].…”

Section: Introductionmentioning

confidence: 99%

“…This system was specifically designed for on-site fluorescent enzyme assays for E. coli. A field portable sample pre-concentration and lysing procedure was applied in the system with a continuous 6-CMUG based assay [24]. The system lysing procedure and assay were tested both in the laboratory and the field.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

ColiSense, today's sample today: A rapid on-site detection of β-d-Glucuronidase activity in surface water as a surrogate for E. coli

Heery

Briciu-Burghina

Zhang

et al. 2016

Talanta

Self Cite

View full text Add to dashboard Cite

ab s t r a c t A sensitive field-portable fluorimeter with incubating capability and triplicate sample chambers was designed and built. The system was optimised for the on-site analysis of E. coli in recreational waters using fluorescent based enzyme assays. The target analyte was β-D-Glucuronidase (GUS) which hydrolyses a synthetic substrate 6-Chloro-4-Methyl-Umbelliferyl-β-D-Glucuronide (6-CMUG) to release the fluorescent molecule 6-Chloro-4-Methyl-Umbelliferyl (6-CMU). The system was calibrated with 6-CMU standards. A LOD of 5 nM and a resolution of less than 1 nM was determined while enzyme kinetic tests showed detection of activities below 1 pmol min 1 mL 1 of sample. A field portable sample preparation, enzyme extraction protocol and continuous assay were applied with the system to analyse freshwater and marine samples. Results from a one day field trial are shown which demonstrated the ability of the system to deliver results on-site within a 75 min period.

show abstract

Section: Introductionmentioning

confidence: 85%

Section: Environmental Sample Testingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…The amount of fluorophore released in a certain period of time is directly proportional to the number of E. coli cells trapped. The assay performs optimally at 44 °C and at pH 6.8 [24].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

ColiSense, today's sample today: A rapid on-site detection of β-d-Glucuronidase activity in surface water as a surrogate for E. coli

Heery

Briciu-Burghina

Zhang

et al. 2016

Talanta

Self Cite

View full text Add to dashboard Cite

show abstract

Microbial enzymatic assays in environmental water samples: Impact of inner filter effect and substrate concentrations

Urvoy

Labry

Delmas

et al. 2020

Limnology & Ocean Methods

View full text Add to dashboard Cite

As microbial enzymatic activities initiate the mineralization of organic matter through the microbial loop, it is important to correctly measure those activities and be able to perform inter-study comparisons. Enzymatic activity assays are typically carried out using fluorogenic substrate analogs, such as 4-Methylumbelliferone and 7-Amino-4-methylcoumarin linked to sugar monomers, phosphate group or amino acids. However, methodological divergences can be found in aquatic science literature, potentially leading to misestimated activities. To highlight some of those methodological key points, we first addressed the potential occurrence of an Inner Filter Effect (IFE), a fluorometric artifact that affects the relationship between fluorophore concentration and fluorescence intensity, due to absorption of exciting or emitted light. It has never been considered in the context of environmental waterstudies before, despite significantly affecting measured activities. IFE occurred with 2 out of 3 tested spectrofluorometers when assaying proteases, although no IFE was detected for phosphatase assays. We also evaluated how substrate concentration ranges might affect kinetic parameters estimation, revealing that a many existing studies might use insufficient maximum substrate concentration. Finally, for single substrate concentration assays, we argued for the use of saturating substrate concentration, as naturally occurring substrates might compete with the fluorogenic analog at trace level. The amendment of a molecule mimicking natural substrates generated a significant inhibition of natural seawater phosphatases and proteases assayed with trace concentrations of fluorogenic substrate, while almost no inhibition occurred at higher concentrations. Those key points need to be addressed in order to assess enzymatic rates and allow inter-study comparison.

show abstract

A Reusable Column Method Using Glycopolymer‐Functionalized Resins for Capture–Detection of Proteins and Escherichia coli

Ajish

Abraham

Subramanian

et al. 2020

Macromolecular Bioscience

View full text Add to dashboard Cite

The use of glycopolymer‐functionalized resins (Resin–Glc), as a solid support, in column mode for bacterial/protein capture and quantification is explored. The Resin–Glc is synthesized from commercially available chloromethylated polystyrene resin and glycopolymer, and is characterized by fourier transform infrared spectroscopy, thermogravimetry, and elemental analysis. The percentage of glycopolymer functionalized on Resin–Glc is accounted to be 5 wt%. The ability of Resin–Glc to selectively capture lectin, Concanavalin A, over Peanut Agglutinin, reversibly, is demonstrated for six cycles of experiments. The bacterial sequestration study using SYBR (Synergy Brands, Inc.) Green I tagged Escherichia coli/Staphylococcus aureus reveals the ability of Resin–Glc to selectively capture E. coli over S. aureus. The quantification of captured cells in the column is carried out by enzymatic colorimetric assay using methylumbelliferyl glucuronide as the substrate. The E. coli capture studies reveal a consistent capture efficiency of 105 CFU (Colony Forming Units) g−1 over six cycles. Studies with spiked tap water samples show satisfactory results for E. coli cell densities ranging from 102 to 107 CFU mL−1. The method portrayed can serve as a basis for the development of a reusable solid support in capture and detection of proteins and bacteria.

show abstract

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates

Cited by 35 publications

References 44 publications

ColiSense, today's sample today: A rapid on-site detection of β-d-Glucuronidase activity in surface water as a surrogate for E. coli

ColiSense, today's sample today: A rapid on-site detection of β-d-Glucuronidase activity in surface water as a surrogate for E. coli

Microbial enzymatic assays in environmental water samples: Impact of inner filter effect and substrate concentrations

A Reusable Column Method Using Glycopolymer‐Functionalized Resins for Capture–Detection of Proteins and Escherichia coli

Contact Info

Product

Resources

About